Mechanistic insights into Enterocin C targeting the undecaprenyl phosphate recycling protein BacA

Enterococcus faecalis is an important opportunistic pathogen responsible for healthcare-associated infections. It is intrinsically resistant to various antibiotics, particularly to cephalosporins and vancomycin, creating an urgent need for alternative therapeutics. In this context, bacteriocins warrant investigations as a potential source of medical-use antibiotics. Herein, we demonstrate that Enterocin C, a class IIb two-peptide bacteriocin, specifically targets the membrane embedded undecaprenyl phosphate recycling protein BacA from enterococci as a cell surface receptor. Using biochemical and biophysical methods, supported by AlphaFold2 modelling and mutagenesis, we deciphered the EntCs molecular interaction pattern with its target, marking the first mechanistic insight of a two-peptide bacteriocin. The two peptides act cooperatively at nanomolar concentrations to interact with the outward-open catalytic pocket of BacA: the peptide EntC1 docks deeply into the catalytic site, inhibits BacAs enzymatic activity and enables the binding of peptide EntC2, eliciting membrane permeabilization, eventually leading to cell death. A comparative analysis with LcnG, a homologous bacteriocin, reveals a conserved interaction pattern, paving the way for bioengineering of these bacteriocins and developing tailored antimicrobial strategies.